Several publications are referenced in this application by numerals in parentheses in order to more fully describe the state of the art to which this invention pertains. Full citations for these references are found at the end of the specification. The disclosure of each of these publications is incorporated by reference herein.
Spider silks comprise a model system for exploring the relationship between the amino acid composition of a protein, the structural properties that result from variations in the amino acid composition of a protein, and how such variations impact protein function. While silk production has evolved multiple times within arthropods, silk use is most highly developed in spiders. Spiders are unique in their lifelong ability to spin an array of different silk proteins (or fibroin proteins) and the degree to which they depend on this ability. There are over 34,000 described species of Araneae (1). Each species utilizes silk, and some ecribellate orb-weavers (Araneoidea) have a varied toolkit of task-specific silks with divergent mechanical properties (2). Araneoid major ampullate silk, the primary dragline, is extremely tough. Minor ampullate silk, used in web construction, has high tensile strength. An orb-web's capture spiral, in part composed of flagelliform silk, is elastic and can triple in length before breaking (3). Each of these fibers is composed of one or more proteins encoded by the spider silk fibroin gene family (4). Sequencing of araneoid fibroins has revealed that these fibroins are dominated by iterations of four simple amino acid motifs: poly-alanine (An), alternating glycine and alanine (GA), GGX (where X represents a small subset of amino acids), and GPG(X)n (5).
Spiders draw fibers from dissolved fibroin proteins that are stored in specialized sets of abdominal glands. Each type of silk is secreted and stored by a different abdominal gland until extruded by tiny spigots on the spinnerets. Spiders use fibroin proteins singly or in combination for a variety of different purposes, including: draglines, retreats, egg sacs, and prey-catching snares. Given these specialized applications, individual silks appear to have evolved to possess mechanical properties (e.g., tensile strength and flexibility) that optimize their utility for particular applications.
Orb web spiders like Nephila are known to produce spider silk proteins derived from several types of silk synthetic glands and are designated according to their organ of origin. Spider silk proteins known to exist include: major ampullate spider proteins (MaSp), minor ampullate spider proteins (MiSp), and flagelliform (Flag), tubuliform, aggregate, aciniform, and pyriform spider silk proteins. Spider silk proteins derived from each organ are generally distinguishable from those derived from other synthetic organs by virtue of-their physical and chemical properties, which render them well suited to different uses. Tubuliform silk, for example, is used in the outer layers of egg-sacs, whereas aciniform silk is involved in wrapping prey and pyriform silk is laid down as the attachment disk.
Most molecular and structural investigations of spider silks have focused on dragline silk, which has an extraordinarily high tensile strength (e.g. Xu & Lewis, Proc. Natl. Acad. Sci., USA 87, 7120-7124, 1990; Hinman & Lewis, J. Biol. Chem. 267, 19320-19324, 1992; Thiel et al., Biopolymers 34, 1089-1097, 1994; Simmons et al., Science 271, 84-87, 1996; Kümmerlen et al., Macromol. 29, 2920-2928, 1996; and Osaki, Nature 384, 419, 1996). Dragline silk, often referred to as major ampullate silk because it is produced by the major ampullate glands, has a high tensile strength (5×109 Nm−2) similar to Kevlar (4×109 Nm−2) (Gosline et al., Endeavour 10, 37-43, 1986; Stauffer et al., J. Arachnol. 22, 5-11, 1994). In addition to this exceptional strength, dragline silk also exhibits substantial (˜35%) elasticity (Gosline et al., Endeavour 10, 37-43, 1986). Thus a structure/function analysis of dragline silk is revealing in terms of the features of a protein which confer strength and elasticity.
Silk strength is widely attributed to crystalline beta-sheet structures. Such protein domains are found in both lepidopteran silks (e.g. Bombyx mori, Mita et al., J. Mol. Evol. 38, 583-592, 1994) and spider silks (Xu & Lewis, Proc. Natl. Acad. Sci., USA 87, 7120-7124, 1990; Hinman & Lewis, J. Biol. Chem. 267, 19320-19324, 1992; Gosline et al., Endeavour 10, 37-43, 1986). In contrast, elasticity is generally thought to involve amorphous regions (Wainwright et al., Mechanical design in organisms, Princeton University Press, Princeton, 1982). More precise characterization of these amorphous components can be revealed by molecular sequence data.
Based on the protein sequences of major ampullate silk proteins, a beta-turn structure was suggested to be the likely mechanism of elasticity (Hinman & Lewis, J. Biol. Chem. 267, 19320-19324, 1992). Assessing this proposition, however, was problematic because dragline silk is a hybrid of at least two distinct proteins which impart both strength and moderate elasticity.
Nephila minor ampullate silk can be distinguished from Nephila major ampullate silk by both physical and chemical properties. On a basic level, the amino acid composition of solubilized minor ampullate silk differs from that of solubilized major ampullate silk. Like the major ampullate silk proteins (major spidroin 1, MaSP1; major spidroin 2, MaSP2), the proteins comprising minor ampullate silk (minor spindroin 1, MiSP1; minor spindroin 2, MiSP2) have a primary structure dominated by imperfect repetition of a short sequence of amino acids. Moreover, in contrast to the elasticity exhibited by major ampullate silk, minor ampullate silk yields without recoil. Minor ampullate silk will stretch to about 25% of its initial length before breaking, thereby exhibiting a tensile strength of nearly 100,000 pounds per square inch (psi). The minor ampullate silk proteins, therefore, exhibit comparatively lower tensile strength and elasticity relative to major ampullate silk proteins.
The capture spiral, on the other hand, is formed from silk proteins derived from the flagelliform and aggregate silk glands. The capture spiral of an orb-web comprises a structure having significant ability to stretch, as would be anticipated for a structure that must capture and retain prey. The capture thread has a lower tensile strength (1×109 Nm−2) but several times the elasticity (>200%) of dragline silk (Vollrath & Edmonds, Nature 340, 305-307, 1989; Kohler & Vollrath, J. Exp. Zool. 271, 1-17, 1995). The flagelliform silk comprises the core fiber of the spiral, while aggregate silk provides a non-fibrous, aqueous coating. Thus, while aggregate silk is an integral part of the elastic capture spiral, it is flagelliform silk that provides the ability to stretch.